Iron–sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in Escherichiacoli

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron–sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron–sulfur cluster biogenesis in E. coli.

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis.However, the proposed ironbound Fur had not been identified in any bacteria.In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box.Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content.Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro.While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF.Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.
The ferric uptake regulator (Fur) is the founding member of the Fur superfamily that regulates intracellular metal homeostasis in bacteria (1,2).In Escherichia coli, Fur regulates over 158 genes (3)(4)(5) involved in intracellular iron homeostasis, energy metabolism, DNA synthesis, oxidative stress response, and bacterial virulence (6,7).In the past 3 decades, it was postulated that when intracellular free iron content is elevated, Fur binds its corepressor a mononuclear ferrous iron (1,2,8,9) and regulates the expression of its target genes by binding to a specific DNA sequence known as the Fur-box (10)(11)(12).However, there is no concrete evidence supporting the idea that Fur binds ferrous iron in E. coli or any other bacteria.A number of Fur crystal structures including E. coli Fur (13), Mycobacterium tuberculosis Fur (14), Vibrio cholerae Fur (15), Helicobacter pylori Fur (16), Campylobacter jejuni Fur (17), and Francisella tularensis Fur (18) have been reported, and none of these structures contains iron.Fur proteins exist as a dimer or tetramer (19), and each Fur monomer has three putative metal binding sites (13,15): site 1 is located within the dimerization domain; site 2 connects the DNA binding domain and the dimerization domain, and site 3 is positioned at the Cterminal end of the dimerization domain.In purified Fur proteins, site 1 and site 2 are often occupied by zinc (15,20).Interestingly, the in vitro studies have showed that E. coli Fur has a relatively weak binding activity for ferrous iron with dissociation constants ranging from 1.2 μM to 55 μM (21,22).However, since the intracellular free iron concentration in E. coli cells grown in LB medium is estimated to be less than 1 μM (23), it may explain why Fur does not bind any ferrous iron in E. coli cells.
Serendipitously, we have found that E. coli Fur binds a [2Fe-2S] cluster in E. coli cells in which intracellular free iron content is elevated (24)(25)(26).A suite of biophysical techniques including the electron paramagnetic resonance and Mössbauer spectroscopy have been used to assign a [2Fe-2S] cluster binding in Fur in E. coli cells in response to elevation of intracellular iron content (25).Additional studies have revealed that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for the Fur-box (24).Furthermore, binding of the [2Fe-2S] cluster in Fur is highly conserved, as Fur proteins from Haemophilus influenzae, V. cholerae, and H. pylori also bind a [2Fe-2S] cluster in E. coli cells with an elevated intracellular free iron content (27).The results suggest that E. coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and regulates intracellular iron homeostasis (24)(25)(26).
In E. coli and many other bacteria, iron-sulfur clusters in proteins are assembled by two iron-sulfur cluster assembly systems: the housekeeping isc operon (iscSUA-hscBA-fdx) (28) and the alternative suf operon (sufABCDSE) (29,30).While the expression of the suf operon is induced under iron starvation (30) or oxidative stress (31,32) conditions, the housekeeping isc operon is mainly responsible for iron-sulfur cluster assembly in cells under normal growth conditions (28).Among the core iron-sulfur cluster assembly proteins encoded by the housekeeping isc operon, IscS is a cysteine desulfurase that catalyzes desulfurization of L-cysteine and delivers sulfur for iron-sulfur cluster assembly in IscU (33)(34)(35)(36).IscU is a scaffold protein that assembles iron-sulfur clusters and transfers the assembled clusters to target proteins (37)(38)(39)(40).IscA was initially characterized as an alternative scaffold protein (41).However, unlike IscU, IscA has a very strong iron binding activity and may act as an iron chaperone for the [4Fe-4S] cluster assembly in proteins (42)(43)(44)(45)(46). Previously, we have found that deletion of IscA and its paralog SufA elevates intracellular free iron content and promotes the [2Fe-2S] cluster assembly in Fur in E. coli cells (25), indicating that IscA is not directly involved in the [2Fe-2S] cluster assembly in Fur.
Here we report that deletion of the iron-sulfur cluster assembly scaffold protein IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content.The in vitro studies further show that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity.While IscU is also required for the [2Fe-2S] cluster assembly in the H. influenzae Fur expressed in E. coli cells, deletion of IscU has very little or no effects on the [2Fe-2S] cluster assembly in the E. coli ferredoxin (47,48) and siderophore reductase FhuF (49,50), indicating that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur.The interplay between regulation of intracellular iron homeostasis and iron-sulfur cluster biogenesis in E. coli cells will be discussed.

Results
The [2Fe-2S] cluster assembly in the Fur requires the ironsulfur cluster assembly scaffold IscU in E. coli cells To test whether the [2Fe-2S] cluster in Fur is assembled by the isc operon in E. coli cells, we have focused on the core iron-sulfur cluster assembly proteins: IscS, IscU, and IscA.IscS is required not only for iron-sulfur cluster biogenesis (33)(34)(35) but also for other key physiological processes such as tRNA modification (34) and biosynthesis of thiamine and NAD (51).Thus, deletion of IscS will affect multiple cellular functions (33).IscA is apparently not required for the [2Fe-2S] cluster assembly in Fur because deletion of IscA and its paralog SufA promotes the [2Fe-2S] cluster assembly in Fur in E. coli cells grown in LB medium (25).Therefore, we have constructed an E. coli mutant in which gene iscU is in-frame deleted using the single-step gene inactivation procedure (52).Deletion of gene iscU was confirmed by PCR (Fig. 1A).As reported previously by other groups (53,54), deletion of IscU has only a mild effect on cell growth of E. coli in LB or M9 medium.
The plasmid encoding E. coli Fur ( 25) is then introduced into E. coli wildtype and the ΔiscU mutant cells.Both cells are grown in M9 medium supplemented with 2 μM iron (ferrous ammonium sulfate) under aerobic growth conditions (24).Wavelength (nm) Absorbance (A) Figure 1.The iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur in E. coli cells.A, PCR confirmation of gene iscU deletion in E. coli cells.Gene iscU was deleted using the single-step inactivation procedure (52) as described previously (43).Two primers (primer 1, 5 0 -TGACCTTTCTCCGCTGTGGG-3 0 , and primer 2, 5 0 -CTTTACCGCGGTTAGCCAGA-3 0 ) were used for the PCR confirmation of the gene iscU deletion.Lane M, 100 bp DNA ladder (NEB).Lane 1, from wildtype E. coli cells.Lane 2, from the ΔiscU mutant cells.B, UV-Vis absorption spectra of Fur proteins purified from wildtype E. coli cells (spectrum 1) and the ΔiscU mutant cells (spectrum 2) grown in M9 medium supplemented with Fe(NH 4 ) 2 (SO 4 ) 2 (2 μM) under aerobic growth conditions.Purified Fur proteins (50 μM) were dissolved in buffer containing NaCl (500 mM) and Tris (20 mM, pH 8.0).Insert (a), the photograph of purified Fur from wildtype E. coli cells (1) and the ΔiscU mutant cells (2).Insert (b), the SDS-PAGE gel of Fur proteins purified from wildtype E. coli cells (lane 1) and the ΔiscU mutant cells (lane 2) grown in M9 medium supplemented with Fe(NH 4 ) 2 (SO 4 ) 2 (2.0 μM) (lane 2).Lane M, the PAGE-MASTER protein markers (GenScript co.) with molecular weights.C, the [2Fe-2S] cluster binding of Fur in E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with increasing concentrations of iron.Fur protein was purified from E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with indicated concentrations of Fe(NH 4 ) 2 (SO 4 ) 2 .The concentration of the [2Fe-2S] cluster in Fur was determined using an extinction coefficient of 10 mM −1 cm −1 at 410 nm (25).The [2Fe-2S] cluster occupancies of Fur were calculated from the ratio of the [2Fe-2S] cluster to Fur and plotted as a function of iron concentrations in M9 medium.Data represent mean ± SD (standard deviation) from three independent experiments.Fur, ferric uptake regulator.

IscU is required for Fur activation
Figure 1B shows that the E. coli Fur purified from wildtype E. coli cells has a red color ((insert a) in Fig. 1B) and three distinct absorption peaks at 325 nm, 410 nm, and 450 nm of the [2Fe-2S] cluster (spectrum 1), as reported previously (24).In contrast, the E. coli Fur purified from the ΔiscU mutant cells grown in M9 medium supplemented with 2 μM iron has no color and very small absorption peaks at 325 nm, 410 nm, and 450 nm (Fig. 1B, spectrum 2).The iron and sulfur content analyses of the purified Fur proteins confirmed that Fur purified from the ΔiscU mutant cells contains very little or no iron-sulfur clusters.As reported previously, increase of the iron concentration in M9 medium to 10 μM does not increase the [2Fe-2S] cluster binding in the Fur in wildtype E. coli cells (Fig. 1C), indicating that 2 μM iron in M9 medium is sufficient to saturate the [2Fe-2S] cluster binding in Fur in wildtype E. coli cells (24).In parallel experiments, Fur expressed in the ΔiscU mutant cells remains as apo-form even when M9 medium is supplemented with 10 μM iron (Fig. 1C).Thus, IscU is essential for the [2Fe-2S] cluster assembly in Fur in E. coli cells in response to elevation of intracellular free iron content.
Fur purified from the E. coli ΔiscU mutant cells fails to bind the Fur-box Upon binding a [2Fe-2S] cluster, E. coli Fur becomes active to bind a specific DNA sequence known as the Fur-box (24).To explore the Fur-box binding activity of Fur purified from the E. coli ΔiscU mutant cells, we utilized the restriction site protection assay as described previously (24).The promoter region of the operon iucABCD which encodes the enzymes for biosynthesis of siderophore aerobactin has a consensus Furbox sequence (5 0 -GAGAATCATTAGCATTCGC-3 0 ) that also contains the restriction hinfI site (5 0 -GANTC-3 0 ) (9).The promoter region of the operon iucABCD was cloned into plasmid pUC19 to create pUC19-iuc as described previously (24).Binding of Fur to the Fur-box protects the hinfI site from being cleaved by HinfI (9) and creates a new DNA band of 787 bp (24).
For the restriction site protection assays, pUC19-iuc is preincubated with increasing concentrations of Fur purified from wildtype E. coli cells, followed by the HinfI digestion.As shown in Figure 2A, the hinfI restriction site of the Fur-box site is protected by the Fur purified from wildtype E. coli cells.In contrast, when pUC19-iuc is preincubated with increasing concentrations of the Fur purified from the E. coli ΔiscU mutant cells, followed by the HinfI digestion, very little or no protection of the hinfI restriction site is observed (Fig. 2A).The intensities of the DNA band of 787 bp on the agarose gel are quantified using ImageJ (NIH) and plotted as a function of the Fur concentrations (Fig. 2B).The results demonstrate that Fur purified from the E. coli ΔiscU mutant cells lost its the Fur-box binding activity.
Fur fails to repress its target gene in the E. coli ΔiscU mutant cells in response to elevation of intracellular free iron content

If deletion of IscU inhibits the [2Fe-2S] cluster assembly in
Fur in E. coli cells (Fig. 1) and produces apo-form Fur that does not bind the Fur-box (Fig. 2), we expect that Fur may not be able to repress its target genes in the E. coli ΔiscU mutant cells in response to elevation of intracellular free iron content.To test this idea, we constructed a reporter gene fur::gfp encoding GFP (green fluorescent protein) controlled by a Furrepressible promoter and inserted the reporter gene into plasmid pBAD (Fig. 3A), as described in Experimental Procedures.The constructed plasmid is introduced into E. coli wildtype and the ΔiscU mutant cells.The cells are grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM iron, respectively.After 5 h of cell growth at 37 C under aerobic growth conditions, the cells are subjected to the GFP fluorescence measurements at 507 nm using the excitation wavelength at 481 nm (55).Relative GFP fluorescence intensities are calculated from the ratios of the fluorescence intensity to the cell density at 600 nm. Figure 3B shows that the expression of the reporter gene fur::gfp is repressed in wildtype E. coli cells grown in M9 medium supplemented with increasing concentrations of iron.About 2 μM iron in M9 medium is sufficient to repress the expression of the reporter gene fur::gfp (Fig. 3B), in agreement with the [2Fe-2S] cluster binding in Fur in wildtype E. coli cells under the same growth conditions (Fig. 1C).In contrast, the expression of the reporter gene fur::gfp remains essentially the same in the E. coli ΔiscU mutant cells grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM iron (Fig. 3B), suggesting that Fur fails to repress the expression of the reporter gene fur::gfp in the E. coli mutant cells with deletion of IscU in response to elevation of intracellular free iron content.
If deletion of gene iscU results in Fur protein that cannot be activated in response to elevation of intracellular free iron content, it is conceivable that the ΔiscU mutant cells would become more sensitive to iron than wildtype cells.To test this idea, both wildtype E. coli and the ΔiscU mutant cells are grown in M9 medium supplemented with increasing concentrations of iron (0-10 μM) under aerobic growth conditions.Figure 3C shows that the cell growth of wildtype E. coli increases when M9 medium is supplemented with iron, likely because M9 medium is iron deficient (0.05 μM total iron) (56).In contrast, the cell growth of the ΔiscU mutant cells A, a reporter gene fur::gfp was constructed and cloned into a plasmid pBAD as described in the Experimental Procedures.An active Fur represses the expression of the reporter gene fur::gfp and decreases the GFP fluorescence in E. coli cells.B, the repressor activity of Fur in E. coli wildtype and the ΔiscU mutant cells.The constructed plasmid containing the reporter gene fur::gfp was introduced into wildtype and the ΔiscU mutant cells.Wildtype E. coli and the ΔiscU mutant cells were grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM Fe(NH 4 ) 2 (SO 4 ) 2. After 5 h growth at 37 o C under aerobic conditions, the cells were subjected to the GFP fluorescence measurements.The relative GFP intensities were obtained from the ratios of the GFP fluorescence intensity to the cell density (A at 600 nm) of the culture.The E. coli cells without the reporter gene fur::gfp were used as the controls.The data represent mean ± SD (standard deviation) from three independent experiments.C, deletion of gene iscU increases the sensitivity of E. coli cells to iron in M9 medium.Wildtype E. coli and the ΔiscU mutant cells were grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM Fe(NH 4 ) 2 (SO 4 ) 2. After 5 h growth at 37 C under aerobic conditions, the cell growth was measured at 600 nm.The ratios of the cell growth in M9 medium supplemented with increasing iron concentrations to the cell growth in M9 medium with no iron addition were plotted as a function of the iron concentrations in M9 medium.The A values at 600 nm (cell growth) of wildtype and the ΔiscU mutant grown in M9 medium with no iron addition were 0.74 ± 0.03 and 0.66 ± 0.04 for, respectively.The data represent mean ± SD (standard deviation) from three independent experiments.Fur, ferric uptake regulator; GFP, green fluorescent protein.
significantly decreases when M9 medium is supplemented with iron.The results further suggest that the E. coli mutant with deletion of gene iscU has an unresponsive Fur and becomes sensitive to excess iron in M9 medium.

IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur in vitro
For many iron-sulfur proteins, iron-sulfur clusters may be readily reconstituted with ferrous iron and sulfide in the presence of thiol-reducing agents such as dithiothreitol in vitro (57).However, we were unable to reconstitute the [2Fe-2S] cluster in apo-form E. coli Fur in vitro, even with excess amounts of ferrous iron and sulfide (25).Since IscU is required for the [2Fe-2S] cluster assembly in Fur in E. coli cells (Fig. 1), we reason that IscU may promote the [2Fe-2S] cluster assembly in E. coli Fur in vitro.
Apo-form Fur (30 μM) (purified from the E. coli ΔiscU mutant cells) is incubated with ferrous ammonium sulfate (1 mM), L-cysteine (1 mM), cysteine desulfurase (IscS) (1 μM), and dithiothreitol (4 mM), with or without IscU (50 μM) at 37 C for 20 min in a cuvette.After incubation, the samples are subjected to UV-Vis absorption measurements.Figure 4A shows that without IscU in the reconstitution solution, very little or no [2Fe-2S] cluster is assembled in apo-form Fur (Spectrum 1), as reported previously (25).However, when IscU  IscU is required for Fur activation is included in the reconstitution solution, the [2Fe-2S] clusters are assembled in apo-form Fur (spectrum 3).Attempts to repurify Fur from the reconstitution solution were not successful as IscU and Fur have similar molecular weights (15.3 kDa and 16.8 kDa, respectively) and similar elusion profiles from the Mono-S column (GE HealthCare co) (data not shown).While addition of a new tag is possible for repurification of Fur from the reaction solution, tags such as glutathione-Stransferase (26 kDa) or maltose binding protein (42 kDa) are too big relative to Fur and IscU and could potentially interfere with the activity of IscU and Fur.Thus, we digitally subtracted the spectrum of IscU after the reconstitution (spectrum 2) from the spectrum of apo-form Fur and IscU after the reconstitution (spectrum 3). Figure 4B shows the UV-Vis absorption spectra of apo-form Fur after the reconstitution without IscU (spectrum 1) and with IscU (spectrum 2), indicating that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur in the presence of ferrous iron, L-cysteine, and IscS in vitro.
Importantly, while apo-form Fur after the reconstitution without IscU remains inactive to bind the Fur-box (lanes 1-3), apo-form Fur after the reconstitution with IscU becomes active to bind the Fur-box (lanes 4-6).As a control, IscU alone has no Fur-box binding activity (Fig. 4C, lane 8).The intensities of the DNA band of 787 bp on the agarose gels are quantified using ImageJ (NIH) and plotted as a function of the Fur concentrations (Fig. 4D).Taken together, the results suggest that the Furbox binding activity of Fur is restored after the [2Fe-2S] cluster is reconstituted in apo-form Fur with IscU in vitro.
IscU is also required for the [2Fe-2S] cluster assembly in the H. influenzae Fur, but not for the [2Fe-2S] cluster assembly in the E. coli ferredoxin and FhuF in E. coli cells To explore whether IscU is also involved in the [2Fe-2S] cluster assembly in other Fur proteins, we expressed the H. influenzae Fur (HI-Fur) in E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with 2 μM iron under aerobic growth conditions.Figure 5A shows that while HI-Fur purified from wildtype E. coli cells has three absorption peaks at 325 nm, 410 nm, and 450 nm of the [2Fe-2S] cluster (Spectrum 1), the HI-Fur purified from the E. coli ΔiscU mutant cells has only very small absorption peaks at 325 nm, 410 nm, and 450 nm (spectrum 2), indicating that the [2Fe-2S] cluster assembly in HI-Fur in E. coli cells also requires IscU.
In parallel, the E. coli ferredoxin, a [2Fe-2S] cluster-containing protein involved in iron-sulfur cluster biogenesis (47,48), is expressed in E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with 2 μM iron under aerobic growth conditions.Figure 5B shows that the UV-Vis absorption spectra of ferredoxin purified from wildtype E. coli cells and the ΔiscU mutant cells are essentially identical.Similarly, deletion of IscU has very little or no effects on the [2Fe-2S] cluster assembly in the E. coli siderophore-reductase FhuF (49, 50) (Fig. 5C).Thus, IscU seems to have a unique role for the [2Fe-2S] cluster assembly in Fur in E. coli cells.

Discussion
Here, we report that the [2Fe-2S] cluster assembly in Fur requires the iron-sulfur cluster assembly scaffold protein IscU, as deletion of gene iscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content.The in vitro studies further suggest that IscU can promote the [2Fe-2S] cluster assembly in apo-form Fur and restore its Furbox binding activity.The results demonstrate that the [2Fe-2S] cluster in Fur is enzymatically assembled by the iron-sulfur cluster assembly system and that IscU has a crucial role for activation of Fur in E. coli cells.
In eukaryotes, iron-sulfur cluster binding to key transcriptional or posttranscriptional regulators mediates intracellular iron sensing.In yeast, paralogs Aft1 and Aft2 activate the iron regulon when iron levels are low (58,59).When iron levels are high, [2Fe-2S] cluster bound Aft1/2 is exported from the nucleus, and the iron regulon is no longer activated (60).In animals, cellular iron homeostasis is regulated posttranscriptionally by iron regulatory protein IRP1 (61,62).When iron is scarce, IRP1 binds to stem-loop structures on mRNA called iron responsive elements to modulate the translation of iron homeostatic genes.When iron is replete, IRP1 binds a [4Fe-4S] cluster and functions as a cytosolic aconitase rather than an IRP (61,62).Our work extends the paradigm of sensing iron through iron-sulfur clusters to bacteria, where we previously demonstrated that Fur binds a [2Fe-2S] cluster, not mononuclear ferrous iron as was commonly thought (1,2,8,9).Moreover, we now also demonstrate that IscU, but not other iron-sulfur cluster assembly proteins like IscA or SufA, is required for [2Fe-2S] cluster assembly in Fur and that IscU plays a privileged role in activating Fur given that other iron-sulfur proteins like FhuF are not dependent on IscU (Fig. 5).
IscU has been shown to interact with cysteine desulfurase IscS and deliver the assembled iron-sulfur cluster to target proteins (37)(38)(39)(40).In eukaryotic cells, deletion of IscU leads to accumulation of iron in mitochondria (63).Using the membrane-permeable iron indicator 2,2 0 -dipyirdyl, we have also found that deletion of IscU elevates the chelatable intracellular iron content in E. coli cells grown in LB medium under aerobic growth conditions (Supplemental Materials).However, unlike the E. coli ΔiscA/ΔsufA mutant cells which also have an elevated intracellular free iron content (25), the E. coli ΔiscU mutant cells fail to assemble the [2Fe-2S] cluster in Fur in E. coli cells in response to elevation of intracellular free iron content (Fig. 1B).The results strongly suggest that the ironsulfur cluster assembly proteins IscA and IscU have their distinct roles in iron-sulfur cluster biogenesis (64).In this context, we propose that while deletion IscA and its paralog SufA increases intracellular free iron content and promotes the [2Fe-2S] cluster assembly in Fur (25), deletion of IscU interrupts the [2Fe-2S] cluster assembly process in Fur and results in apo-form Fur in E. coli cells.It should be pointed out that IscU is not required for the [2Fe-2S] cluster assembly in ferredoxin and FhuF in E. coli cells grown in M9 medium supplemented with 2 μM iron, suggesting that IscU may have a unique role for the [2Fe-2S] cluster assembly in the iron regulatory protein Fur.The underlying mechanism remains to be further investigated.
Ironically, the expression of iron-sulfur cluster assembly systems encoded by the housekeeping isc operon (iscSUA-hscBA-fdx) (28) and the alternative suf operon (sufABCDSE) (29) is also regulated by Fur in E. coli cells.First, an active Fur will bind the Fur-box in the promoter of the suf operon and repress the expression of the operon (30,31).Under iron starvation (30) or oxidative stress conditions (31,32), Fur becomes inactive and dissociates from the Fur-box site, and the suf operon is expressed for iron-sulfur cluster biogenesis or repair (30,31).Second, Fur indirectly regulates the expression of the isc operon via an iron-responsive RNA, RyhB (65,66).RyhB is a small RNA (containing 90 nt) that regulates at least 18 operons and over 56 genes in E. coli (66).The expression of RyhB is repressed by an active Fur (65).When Fur is inactivated, highly expressed RyhB binds to specific mRNA of the isc operon, resulting in inhibition of iron-sulfur cluster assembly in cells (67).Our finding that the [2Fe-2S] cluster in Fur is assembled by IscU reveals a novel link between iron-sulfur cluster biogenesis and regulation of intracellular iron homeostasis in E. coli cells (Fig. 6).
could not be readily reconstituted in apo-form E. coli Fur in vitro even with excess amounts of ferrous iron and sulfide (25).Only when IscU is included in the reconstitution solution, the [2Fe-2S] cluster is assembled in apo-form Fur in the presence of iron and L-cysteine and IscS (Fig. 4).The weak binding activity of Fur for the [2Fe-2S] cluster may reflect its regulatory nature as a sensor of intracellular free iron content.When intracellular free iron content is depleted, Fur does not bind the [2Fe-2S] cluster and is inactive.When intracellular free iron content is elevated, the [2Fe-2S] cluster is enzymatically assembled in Fur, and Fur becomes active to repress the expression of iron uptake systems, the iron-responsive RNA RyhB, and many other target genes (65,66).Repression of RyhB expression will then enhance the expression of the isc operon and promote iron-sulfur cluster biogenesis activity in E. coli cells (67).Thus, regulation of intracellular iron homeostasis and iron-sulfur cluster biogenesis are closely coupled via this global iron regulator Fur in E. coli cells.

HinfI site protection assays
The Fur-box binding activity of E. coli Fur was analyzed using the hinfI site protection assays (9).The Fur-box in the E. coli iucABCD promoter (5 0 -GAGAATCATTAG-CATTCGC-3 0 ) contains the restriction enzyme HinfI site (5 0 -GAATC-3 0 ).Binding of Fur to the Fur-box protects the hinfI site (highlighted) from being cleaved by HinfI (9).The iucABCD promoter was synthesized (GenScript co.) and cloned into plasmid pUC19 via BamHI and HindIII sites to create pUC19-iuc.For the hinfI site protection assays, pUC19iuc was preincubated with purified Fur proteins in 10 μl reaction solutions containing MgCl 2 (2 mM), NaCl (150 mM), bovine serum albumin (0.1 mg/ml), and Tris (20 mM, pH 8.0) for 10 min at room temperature.Restriction enzyme HinfI (0.5 unit) (New England Biolab co.) was then added to the reaction solutions.After incubation at 37 C for 10 min, the reaction was stopped by adding 2 μl 6× loading buffer (New England Biolab co).The digested DNA products were separated on 1.5% agarose electrophoresis gel containing ethidium bromide (0.1 μg/ml) in 0.5× TAE (Tris-acetate-EDTA) buffer, run at 120 V for 35 min.The gel images were taken using the Kodak Gel Logic 200 Imaging System.The intensities of the DNA band on the agarose gels were quantified using ImageJ (NIH).
In vivo repressor activity assays of Fur using the reporter gene Fur::gfp  EcoRV and HindIII sites.The highlighted sequence represents the Fur-box (10,11).The plasmid containing the reporter gene fur::gfp was introduced into wildtype E. coli (MC4100) and the ΔiscU mutant cells.Overnight culture was inoculated (1:100 dilution) in freshly prepared M9 medium supplemented with 20 amino acids (10 μg each amino acid/ml), thiamine (1 μg/ ml), glycerol (0.4%), Fe(NH 4 ) 2 (SO 4 ) 2 (0-10.0μM), and ampicillin (100 μg/ml), and grown at 37 C under aerobic condition for 5 h.Cells were then subjected to the GFP fluorescence measurements at 507 nm using the excitation wavelength at 481 nm in the fluorescence spectrometer (PerkinElmer LS-3).The relative fluorescence intensity was calculated by dividing the green fluorescence intensity with the cell density (A at 600 nm).The E. coli cells without the reporter gene fur::gfp grown in M9 medium supplemented with increasing concentrations of Fe(NH 4 ) 2 (SO 4 ) 2 (0-10.0μM) were used as the controls.

Statistical analysis
All data are expressed as mean ± SD (standard deviation) from at least three independent experiments.

Figure 2 .
Figure2.The Fur-box binding activity of Fur purified from E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with 2 μM iron.A, the restriction site protection assays of Fur.pUC19-iuc (3.2 nM) was preincubated with increasing concentrations of purified Fur proteins, followed by digestion with HinfI (0.5 unit) at 37 C for 10 min.The digested DNA products were separated by 1.5% agarose gel electrophoresis.Lanes 1 to 4, pUC19-iuc (3.2 nM) was preincubated with 2.0 μM, 1.0 μM, 0.5 μM, and 0.25 μM Fur purified from wildtype E. coli cells, respectively.Lanes 5 to 8, pUC19-iuc (3.2 nM) was preincubated with 2.0 μM, 1.0 μM, 0.5 μM, and 0.25 μM Fur purified from the ΔiscU mutant E. coli cells, respectively.Lane 9, no Fur protein was added before the HinfI digestion.Lane 10, pUC19-iuc only with no HinfI digestion.B, relative binding activity of Fur proteins purified from wildtype and the ΔiscU mutant E. coli cells.The intensities of the DNA band of 787 bp shown in (A) were quantified using ImageJ and plotted as a function of the Fur concentrations.Data represent mean ± SD (standard deviation) from three independent experiments.Fur, ferric uptake regulator.

Figure 3 .
Figure 3. Deletion of gene iscU prevents activation of Fur as a repressor in E. coli cells.A, a reporter gene fur::gfp was constructed and cloned into a plasmid pBAD as described in the Experimental Procedures.An active Fur represses the expression of the reporter gene fur::gfp and decreases the GFP fluorescence in E. coli cells.B, the repressor activity of Fur in E. coli wildtype and the ΔiscU mutant cells.The constructed plasmid containing the reporter gene fur::gfp was introduced into wildtype and the ΔiscU mutant cells.Wildtype E. coli and the ΔiscU mutant cells were grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM Fe(NH 4 ) 2 (SO 4 ) 2. After 5 h growth at 37 o C under aerobic conditions, the cells were subjected to the GFP fluorescence measurements.The relative GFP intensities were obtained from the ratios of the GFP fluorescence intensity to the cell density (A at 600 nm) of the culture.The E. coli cells without the reporter gene fur::gfp were used as the controls.The data represent mean ± SD (standard deviation) from three independent experiments.C, deletion of gene iscU increases the sensitivity of E. coli cells to iron in M9 medium.Wildtype E. coli and the ΔiscU mutant cells were grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM Fe(NH 4 ) 2 (SO 4 ) 2. After 5 h growth at 37

Figure 6 .
Figure 6.A proposed model for the interplay between iron-sulfur cluster biogenesis and regulation of intracellular iron homeostasis in E. coli cells.When intracellular free iron content is depleted, IscU cannot assemble a [2Fe-2S] cluster in Fur, and Fur is inactive.When intracellular free iron content is elevated, IscU, together with L-cysteine, cysteine desulfurase (IscS) and other iron-sulfur cluster assembly machinery, assembles a [2Fe-2S] cluster in Fur, and Fur becomes an active repressor to bind the Furbox and regulates intracellular iron homeostasis.A structural zinc binding site is shown in apo-Fur and the [2Fe-2S] cluster-bound Fur.Fur, ferric uptake regulator.